Amines, amides, and thio- and carboxamides of (-)-cytisine as Nfat transcription factor modulators

Article abstract of DOI:10.1007/s10600-014-0996-7

A library of derivatives of the quinolizidine alkaloid (-)-cytisine with amine, amide, and thio- and carboxamides in the 3-, 5-, and 12-positions were synthesized. Their activity with respect to NFAT transcription factor was studied. It was shown that NFAT modulation activity was a function of the nature of the substituent.

Full text of DOI:10.1007/s10600-014-0996-7

Chemistry of Natural Compounds, Vol. 50, No. 3, July, 2014 [Russian original No. 3, May–June, 2014]
AMINES, AMIDES, AND THIO- AND CARBOXAMIDES
OF (–)-CYTISINE AS NFAT TRANSCRIPTION FACTOR
MODULATORS
1
2*
1
Yu. V. Vakhitova, I. P. Tsypysheva, M. Kh. Salimgareeva,
2
2
1
A. V. Kovalꢀskaya, A. N. Lobov, U. Sh. Fatkullina,
L. F. Zainullina, and M. S. Yunusov
1
2
A library of derivatives of the quinolizidine alkaloid (–)-cytisine with amine, amide, and thio- and
carboxamides in the 3-, 5-, and 12-positions were synthesized. Their activity with respect to NFAT transcription
factor was studied. It was shown that NFAT modulation activity was a function of the nature of the substituent.
Keywords: (–)-cytisine, NFAT transcription factor, structure–activity.
The discovery of compounds that modulate transcription factor (TF) activity is currently used to select potential
targeting drug candidates. Numerous studies showed that activation of NFAT (nuclear factor of activated T-cells) TF plays an
important role in the normal functioning of the immune system. However, excessive activation of this TF causes the development
of immunopathological reactions such as autoimmune processes, inflammation, transplant rejection, carcinogenesis, and
metastasis. Thus, the search for new NFAT modulators is extremely critical [1–3].
A sufficient number of NFAT inhibitors/activators of natural and synthetic origin have now been identified [4–6].
However, such studies of the quinolizidine alkaloid (–)-cytisine and its derivatives have not been conducted.
This TF was chosen as the screening target because the anti-inflammatory and immunomodulating activities of several
quinolizidine alkaloids were reported [7, 8].
O
O
N
H
N
H
N
N
N
N
N
9
O
O
f
NH
2
12
NH
7
5
CH
CH
3
3
N
a
a
8
N
N
3
N
9
H N
2
10 1
2
3
O
O
O
O
b orc
d
e
R
S
O
NH
N
CH
H
3
CH
3
N
N
N
H
Ph
N
R
N
O
N
N
N
H
4, 5
7, 8
6
O
O
4: R = Bn; 5: R = AllNH(CS); 7: R = Allyl; 8: R = Ph
a. [9]; b. PhCHO, THF, 64ꢁC, NaBH ; c. AllNCS, C H , 80 °Ñ; d. [11]; e. RNCO, C H , 80ꢁÑ; f. CH (C H NCO) , C H , 80ꢁÑ
4
6
6
6
6
2
6
4
2
6 6
Scheme 1
1) Institute of Biochemistry and Genetics, Ufa Scientific Center, Russian Academy of Sciences, Ufa; 2) Institute of
Organic Chemistry, Ufa Scientific Center, Russian Academy of Sciences, 450054, Ufa, Prosp. Oktyabrya, 71, e-mail:
tsipisheva@anrb.ru. Translated from Khimiya Prirodnykh Soedinenii, No. 3, May–June, 2014, pp. 430–434. Original article
submitted December 12, 2013.
©
498
0009-3130/14/5003-0498 2014 Springer Science+Business Media New York

We synthesized N-benzyl-(12-N-methylcytisine)-3-amine (4), N-(12-methylcytisin-3-yl)-Nꢀ-allylthiourea (5),
N-(cytisino-1-carbonothioyl)benzamide (6), N-(12-methylcytisin-5-yl)-Nꢀ-allylurea (7), N-(12-methylcytisin-5-yl)-Nꢀ-
phenylurea (8), and N,Nꢀ-[methylene-bis(4,1-phenylene)]dicytisinecarboxamide (9) in order to discover (–)-cytisine derivatives
that were modulators of this TF activity (Scheme 1). These supplemented the library of (–)-cytisine (1) derivatives with
variously shaped thio- and carboxamide groups that were prepared by us earlier [9, 10].
Preliminary experiments assessed the potential cytotoxic properties (MTT test) of synthesized 2 and 4–23 that were
comprised by the library of derivatives of 1 with amine, amide, and thio- and carboxamide groups in the 3-, 5-, and
12-positions. As it turned out, none of these compounds exhibited cytotoxic activity with respect to conditionally normal cell
line HEK293 (IC > 1 mM, data not presented).
50
R
2
(CH )
2 6
R
N
N
N
N
N
N
R
1
O
O
O
10 - 22
23
10: R = Me, R = R = H; 11: R = Bn, R = R = H
1
2
1
2
12: R = Bz, R = R = H; 13: R = (CO)NH , R = R = H
1
2
2
1
2
14: R = (CO)NHAll, R = R = H; 15: R = (CO)NHPh, R = R = H
1
2
1
2
16: R = (CO)NHAd, R = R = H; 17: R = (CS)NH , R = R = H
1
2
2
1
2
18: R = (CS)NHAll, R = R = H; 19: R = (CS)NHPh, R = R = H
1
2
1
2
20: R = (CS)NHAd, R = R = H; 21: R = Me, R = NH(CO)NHAll, R = H
1
2
1
2
22: R = Me, R = NH(CO)NHPh, R = H
1
2
Next, we studied the ability of 1, 2, and 4–23 to modulate NFAT TF activity using NFAT-regulated luciferase reporter
constructs. Results for the effects of the synthesized compounds on the DNA-binding activity of NFAT TF are given below.
Compound
IC50/EC50 values
Compound
IC50/EC50 values
IC50 > 1 mM
EC50 0.9 mM
EC50 0.6 mM
IC50 > 1 mM
IC50 0.3 mM
EC50 0.7 mM
IC50 > 1 mM
EC50 1.3 mM
IC50 > 5 mM
IC50 0.39 mM
IC50 0.84 mM
IC50 1 mM.
1
2
18
19
20
21
22
23
4
8
9
17
As it turned out, only several of the library compounds were active with respect to this TF. In general, the compounds
had weak activity with respect to NFAT TF. This was evident in the IC and EC values, which were in the millimolar
50
50
concentration range.
Nevertheless, several trends in the structure–activity relationship could be discerned. These could possibly predict
which new derivatives with more pronounced activity with respect to this TF should be synthesized. Thus, a comparative
quantitative assessment of the effects of the studied compounds on NFAT-dependent luciferase activity showed that the shape
of the substituent did not noticeably affect this parameter whereas the nature of the substituent could play a significant role. In
particular, the IC values for bimolecular carboxamide 9 and allylurea 21 were 0.3 and 0.39 mM, respectively, and characterized
50
them as NFAT binding inhibitors. The activity of this TF was slightly suppressed for 3ꢀ-N-phenylcarboxamide 22 (IC 0.84 mM).
50
Thiocarboxamide 17 (EC 0.7 mM) and 3-amine 2 and its benzyl derivative 4 (EC 0.9 and 0.6 mM, respectively)
50
50
were capable of regulating positively NFAT TF activity.
Thus, derivatives of 1 primarily with a substituted amine in the 3-position of the 2-pyridone ring exhibited modulating
properties with respect to NFAT TF. Thus, carboxamide 8 inhibited NFAT activity whereas starting amine 2 and its benzyl
derivative 4 activated this TF. Inhibiting properties were also found for bimolecular 9; activating, for thiourea 17.
EXPERIMENTAL
The starting material was commercially available (–)-cytisine (CAS 485-35-8). The course of reactions was monitored
®
by TLC on Alugram plates with an aluminum backing coated with a layer (0.2 mm) of standard silica gel 60 (Macherey-
Nagel, Germany). Melting points of crystalline compounds were determined on a Boetius apparatus (PHMK 05, VEB
Wagetechnik Rapido Radebeul). Optical rotation angles were measured on a PerkinElmer 341 LC polarimeter (Na lamp, 589 nm).
499

13
15
PMR, C NMR, and N NMR spectra were recorded relative to TMS internal standard on equipment at the Khimiya
Center for Collective Use, IOC, USC, RAS, i.e., Bruker Avance III pulsed spectrometers at operating frequency 500.13 MHz
1
13
15
1
1
1
13
1
13
1
1
( H), 125.47 MHz ( C), and 50.67 MHz ( N). Two-dimensional H– H COSY, H– C HSQC, H– C HMBC, and H– H
NOESY spectra were recorded using standard multi-pulse sequences of the instrument software. Physicochemical constants
of 2, 3, 6, and 10–23 agreed with the published values [9, 10, 12–18].
N-Benzyl-(12-N-methylcytisine)-3-amine (4). A mixture of amine 2 (0.2 g, 0.9 mmol) and benzaldehyde (0.12 g,
1.1 mmol) in C H (5 mL) was refluxed until 2 disappeared completely (TLC monitoring) and concentrated at reduced pressure.
6
6
The residue was dissolved in MeOH (1 mL), cooled, treated with NaBH (0.45 g, 12.0 mmol), stirred on a magnetic stirrer for
4
1 h, and evaporated. The residue was dissolved in H O and extracted with CHCl (5 ꢂ 10 mL). The extracts were combined,
2
3
dried over Na SO , and evaporated. The residue was chromatographed over SiO (EtOAc eluent) to afford 4 (0.23 g, 80%).
2
4
2
20
–1
Amorphous compound, [ꢃ] –28.0ꢁ (ñ 1.55 CHCl ). IR spectrum (ꢄ, cm ): 3395, 2930, 2778, 1636, 1586, 1547,
1514, 1448, 1380, 1359, 1262, 1162, 1056, 1035, 776, 725. Í NMR spectrum (CDCl , ꢅ, ppm, J/Hz): 1.66 (1H, dtd, J = 12.7,
D
3
1
2
3
3
3
4
2
3
3
4
4
J
= 3.2, J = 3.2, J
= 1.3, H -8), 1.78 (1H, ddt, J = 12.7, J = 3.4, J = 3.4, J
= 1.7, J
= 1.7, H -8), 2.08
8-7
8-9
8-10
anti
8-7
8-9
8-11
8-13 syn
2
3
4
2
3
(3H, s, CH ), 2.17 (1H, ddd, J = 11.3, J
(1H, m, H-9), 2.74 (1H, ddt, J = 10.5, J
= 2.5, J
= 1.0, H -11), 2.16 (1H, dd, J = 10.5, J
= 2.4, H -13), 2.35
-13), 2.80 (1H, m, H-7), 2.85 (1H, ddt,
3
11-9
11-10
4
exo
13-7 exo
2
3
4
= 3.2, J
= 1.7, J
= 1.7, H
-11), 3.94 (1H, ddd, J = 15.1, J
13-7
= 1.7, H
13-11
13-8
endo
3
2
3
4
4
2
4
J = 11.3, J
= 3.3, J
= 1.7, J
= 7.00, J
= 1.0, H -10), 4.12
11-9
11-13
= 1.0, J
11-8
endo
10-9
10-11
exo
2
3
4
3
(1H, dt, J = 15.1,
J
= 1.0, H
-10), 4.28 (2H, s, CH NH), 5.36 (1H, s, NH), 5.85 (1H, d, J = 7.4, H-5),
10-9
10-8
endo
2
5-4
3
6.13 (1H, d, J = 7.4, H-4), 7.22 (2H, t, J = 7.7, 7.7, H-3ꢀ, 5ꢀ), 7.29 (1H, d, J =7.7, H-4ꢀ), 7.33 (2H, d, J = 7.7, H-2ꢀ, 6ꢀ).
4-5
13
C NMR spectrum (CDCl , ꢅ, ppm): 26.1 (C-8), 27.9 (C-9), 34.6 (C-7), 46.3 (C-H ), 47.6 (C-H NH), 50.2 (C-10), 62.2
3
3
2
(C-11), 63.2 (C-13), 104.8 (C-5), 107.5 (C-4), 126.9 (C-4ꢀ), 127.2 (C-2ꢀ, 6ꢀ), 128.4 (C-3ꢀ, 5ꢀ), 136.0 (C-3), 136.0 (C-6), 139.0
15
+
(C-1ꢀ), 157.9 (C-2). N NMR spectrum (CDCl , ꢅ, ppm): 28.93 (N-12), 60.35 (NH), 167.64 (N-1). HR-MS (EI): ꢆÌ
3
325.1716, calcd for C H N O 325.1785.
15 19
3 2
N-(12-Methylcytisin-3-yl)-Nꢀ-allylthiourea (5). A solution of amine 2 (0.2 g, 0.9 mmol) in C H (5 mL) was
6
6
treated with allylisothiocyanate (0.1 mL, 1 mmol), refluxed until 2 disappeared completely (TLC monitoring), and evaporated.
The residue was chromatographed over SiO (CHCl –MeOH eluent, 97:3) to afford 5 (0.26 g, 90%).
2
3
20
–1
Amorphous compound, [ꢃ] –42.0ꢁ (ñ 1.5, CHCl ). IR spectrum (ꢄ, cm ): 3272, 2935, 1635, 1579, 1530, 1341,
1269, 1232, 1150, 1128. Í NMR spectrum (CDCl , ꢅ, ppm, J/Hz): 1.73 (1H, dtd, J = 13.3, J
D
3
1
2
3
3
= 3.4, J = 3.4,
3
8-7
8-9
4
2
3
3
4
4
J
= 1.3, H -8), 1.83 (1H, ddt, J = 13.3, J = 3.4, J = 3.4, J
= 1.7, J
= 1.7, H -8), 2.09 (3H, s, H-14), 2.21
8-10
anti
8-7
8-9
8-11
8-13 syn
2
3
4
2
3
(1H, ddd, J = 11.3, J
= 2.5, J
= 3.1, J
= 1.0, H -11), 2.22 (1H, dd, J = 11.2, J
= 1.7, J
-11), 2.94 (1H, m, H-7), 3.85 (1H, ddd, J = 15.3, J
= 2.4, H -13), 2.36 (1H, m, H-9), 2.80
11-9
11-10
exo
13-7 exo
-13), 2.83 (1H, ddt, J = 11.3, J
2
3
4
4
2
3
4
(1H, ddt, J = 11.2, J
= 1.7, H
= 3.3, J
= 1.7,
13-7
13-11
13-8
endo
11-9
11-13
4
2
3
4
J
= 1.7, H
= 6.6, J
= 1.0, H -10), 3.95 (2H, m, H-1ꢀ),
11-8
endo
10-9
10-11
exo
2
3
4
3
4
3.98 (1H, dt, J = 15.3, J
= 1.0, J
= 1.0, H
-3ꢀ), 5.93 (1H, ddt, J
-10), 5.08 (1H, dq, J
= 10.2, J
= 1.7, H -3ꢀ), 5.24 (1H, dq,
10-9
= 1.9, H
10-8
endo
3
3ꢀ-2ꢀ
3ꢀ-1ꢀ
cis
3
4
3
3
3
J
= 17.0, J
= 17.1, J
= 10.2, J
= 5.9, H-2ꢀ), 6.11 (1H, d, J = 7.8,
3ꢀ-2ꢀ
3ꢀ-1ꢀ
trans
2ꢀ-3ꢀ
2ꢀ-3ꢀ
2ꢀ-1ꢀ
5-4
3
3
13
H-5), 7.52 (1H, t, J
= 5.5, NHCH ), 8.30 (1H, d, J = 7.8, H-4), 8.90 (1H, s, NHCO). C NMR spectrum (CDCl , ꢅ,
NH-1ꢀ
2
4-5
3
ppm): 25.5 (C-8), 27.7 (C-9), 34.6 (C-7), 42.2 (C-1ꢀ), 46.1 (CH ), 50.7 (C-10), 61.9 (C-11), 62.6 (C-13), 106.2 (C-5), 114.8
3
15
(C-3ꢀ), 120.5 (C-4), 128.3 (C-3), 136.4 (C-2ꢀ), 140.7 (C-6), 156.3 (CONH), 157.7 (C-2). N NMR spectrum (CDCl , ꢅ, ppm):
28.98 (N-12), 85.58 (NHCH ), 97.23 (NHCO), 171.85 (N-1). HR-MS (EI): [M] 318.1526, calcd for C H N O 318.1509.
3
+
2
15 19 3 2
20
N-(Cytisino-1-carbonothioyl)benzamide (6) was prepared as before [11], mp 121–122°C (EtOAc), [ꢃ] –318.0°
D
(c 1.93, MeOH).
1
E-conformer: Í NMR spectrum (DMSO-d , ꢅ, ppm, J/Hz): 1.93 (1H, m, H -8), 2.13 (1H, m, H -8), 2.76 (1H, m,
H-9), 3.20 (1H, m, H-7), 3.43 (1H, br.d, J = 13.2, H -11), 3.55 (1H, br.d, J = 13.1, H -13), 3.74 (1H, br.d, J = 15.7,
6
anti
syn
2
2
2
exo
exo
2
2
2
H
-10), 3.93 (1H, br.d, J = 13.1, H
-13), 4.19 (1H, br.d, J = 15.7, H
-10), 5.50 (1H, br.d, J = 13.2, H
-11), 6.20
exo
endo
endo
3
endo
3
3
3
(1H, br.d, J = 7.00, H-5), 6.32 (1H, br.d, J = 9.1, H-3), 7.42 (1H, dd, J = 9.1, J = 7.00, H-4), 7.48 (2H, m, H-3ꢀ,
5-4
3-4
4-3
4-5
13
5ꢀ), 7.61 (1H, m, H-4ꢀ), 7.77 (2H, br.d, J = 7.9, H-2ꢀ, 6ꢀ). C NMR spectrum (DMSO-d , ꢅ, ppm): 24.5 (C-8), 28.1 (C-9), 34.6
6
(C-7), 47.9 (C-10), 54.9 (C-11), 57.2 (C-13), 105.0 (C-5), 116.4 (C-3), 128.2 (C-2ꢀ, 6ꢀ), 128.4 (C-3ꢀ, 5ꢀ), 132.3 (C-1ꢀ), 132.5
(C-4ꢀ), 139.1 (C-4), 148.8 (C-6), 162.5 (CO), 163.9 (C-2), 180.9 (CS).
1
Z-conformer: Í NMR spectrum (DMSO-d , ꢅ, ppm, J/Hz): 1.91 (1H, m, H -8), 2.11 (1H, m, H -8), 2.53 (1H, m,
6
anti
syn
2
2
2
H-9), 3.38 (1H, m, H-7), 3.45 (1H, br.d, J = 13.2, H -11), 3.47 (1H, br.d, J = 13.1, H -13), 3.70 (1H, br.d, J = 15.7,
-10), 3.90 (1H, br.d, J = 15.7, H
(1H, br.d, J = 9.1, H-3), 6.31 (1H, br.d, J = 7.00, H-5), 7.38 (1H, dd, J = 9.1, J = 7.00, H-4), 7.50 (2H, m, H-3ꢀ, 5ꢀ),
exo
exo
2
2
2
H
-10), 4.12 (1H, br.d., J = 13.2, H
-11), 5.35 (1H, br.d, J = 13.1, H
-13), 6.29
exo
endo
endo
endo
3
3
3
3
3-4
5-4
4-3
4-5
13
7.59 (1H, m, H-4ꢀ), 7.91 (2H, br.d, J = 7.9, H-2ꢀ, 6ꢀ).
C NMR spectrum (DMSO-d , ꢅ, ppm): 24.4 (C-8), 27.6 (C-9), 34.2
6
500

(C-7), 48.4 (C-10), 55.6 (C-13), 55.9 (C-11), 105.8 (C-5), 116.1 (C-3), 128.2 (C-2ꢀ, 6ꢀ), 128.4 (C-3ꢀ, 5ꢀ), 132.3 (C-1ꢀ), 132.5
(C-4ꢀ), 139.1 (C-4), 148.6 (C-6), 162.5 (CO), 163.7 (C-2), 181.2 (CS).
N-(12-Methylcytisin-5-yl)-Nꢀ-allylurea (7) was prepared from amine 3 (0.2 g, 0.9 mmol) and allylisocyanate
(0.09 mL, 1 mmol) by the method for 5. Yield of 7, 0.26 g (96%).
20
–1
Amorphous compound, [ꢃ] –30.2ꢁ (ñ 4.2, MeOH). IR spectrum (ꢄ, cm ): 3286, 2931, 2852, 2786, 1656, 1598,
D
1
1548, 1498, 1442, 1365, 1314, 1287, 1229, 1150, 1059, 1028, 833, 756, 694. Í NMR spectrum (CD OD, ꢅ, ppm, J/Hz): 1.82
(1H, dtd, J = 13.3, J = 3.3, J = 3.3, J
3
2
3
3
4
2
3
3
4
= 1.3, H -8), 1.86 (1H, dtt, J = 13.3, J = 3.4, J = 3.4, J
= 1.7,
= 2.5,
8-7
8-9
8-10
anti
8-7
8-9
8-11
4
4
2
3
2
3
J
J
= 1.7, H -8), 2.17 (3H, s, CH ), 2.28 (1H, dd, J = 11.2, J
= 2.4, H -13), 2.31(1H, ddd, J = 11.4, J
8-13
syn
3
13-7 exo
11-9
2
3
4
4
= 1.0, H -11), 2.48 (1H, m, H-9), 2.95 (1H, ddt, J = 11.2, J
= 3.1, J
= 1.7, J
= 1.7, H
-13), 2.97 (1H,
11-10
2
exo
13-7
13-11
13-8
endo
3
4
4
3
4
ddt, J = 11.4, J
= 3.3, J
= 1.7, J
= 1.7, H
-11), 3.33 (1H, m, H-7), 3.77 (2H, dt, J
= 5.5, J
= 1.9,
= 1.0,
-3ꢀ),
11-9
11-13
11-8
endo
= 6.7, J
1ꢀ-2ꢀ
1ꢀ-3ꢀ
4
2
3
4
2
3
J
J
= 1.7, H-1ꢀ), 3.92 (1H, ddd, J = 15.3, J
= 1.0, H -10), 4.00 (1H, dt, J = 15.3, J
1ꢀ-3ꢀ
10-9
4
10-11
exo
10-9
4
3
3
4
= 1.0, H
5.86 (1H, ddt, J
-10), 5.08 (1H, dq, J
= 10.2, J
= 1.7, H -3ꢀ), 5.18 (1H, dq, J
= 17.0, J
= 1.9, H
10-8
endo
3ꢀ-2ꢀ
3ꢀ-1ꢀ
cis
3ꢀ-2ꢀ
3ꢀ-1ꢀ
trans
3
3
3
3
3
3
= 17.0, J
= 10.2, J
= 5.9, J
= 5.9, H-2ꢀ), 6.43 (1H, d, J = 9.2, H-3), 7.41 (1H, d, J = 9.2,
2ꢀ-3ꢀ
2ꢀ-3ꢀ
2ꢀ-1ꢀ
2ꢀ-1ꢀ
3-4
4-3
13
H-4). C NMR spectrum (CD OD, ꢅ, ppm): 25.7 (C-8), 28.9 (C-9), 31.7 (C-7), 43.4 (C-1ꢀ), 46.4 (CH ), 52.1 (C-10), 61.0
3
3
(C-13), 62.9 (C-11), 115.5 (C-3ꢀ), 116.6 (C-3), 117.4 (C-5), 136.7 (C-2ꢀ), 143.4 (C-4), 150.6 (C-6), 160.0 (CO), 164.7 (C-2).
+
HR-MS (EI): [M] 302.1767, calcd for C H N O 302.1723.
15 19
3 2
N-(12-Methylcytisin-5-yl)-Nꢀ-phenylurea (8) was prepared from amine 3 (0.2 g, 0.9 mmol) and phenylisocyanate
(0.11 mL, 1 mmol) by the method for 5. Yield of 8, 0.3 g (98%).
20
–1
Amorphous compound, [ꢃ] –17.5ꢁ (ñ 1.4, MeOH). IR spectrum (ꢄ, cm ): 3282, 2936, 2786, 1652, 1532, 1466,
D
1
1421, 1364, 1315, 1266, 1232, 1148, 1059, 1027, 917, 828, 799, 764. Í NMR spectrum (CD OD, ꢅ, ppm, J/Hz): 1.83 (1H,
dtd, J = 13.3, J = 3.3, J = 3.1, J
3
2
3
3
4
2
3
3
4
= 1.3, H -8), 1.89 (1H, ddt, J = 13.3, J = 3.4, J = 3.4, J
= 1.7,
= 2.5,
8-7
8-9
8 -10
anti
8-7
8-9
2
8-11
4
4
2
3
3
J
J
= 1.7, H -8), 2.16 (3H, s, CH ), 2.28 (1H, dd, J = 11.2, J
= 2.4, H -13), 2.29 (1H, ddd, J = 11.9, J
-13), 2.95 (1H, br.d, J = 11.9, H
= 1.0, H -10), 4.02 (1H, dt, J = 15.3, J
8-13
syn
3
13-7
exo
11-9
2
2
= 1.0, H -11), 2.49 (1H, m, H-9), 2.95 (1H, d, J = 11.2, H
-11), 3.39 (1H, m,
= 1.0, H
11-10
exo
endo
endo
2
3
4
2
3
4
H-7), 3.94 (1H, ddd, J = 15.3, J
= 6.7, J
= 1.0, J
-10),
endo
10-9
10-11
exo
10-9
10-8
3
6.47 (1H, d, J = 9.5, H-3), 7.01 (1H, br.t, J =7.3, H-4ꢀ), 7.27 (2H, dd, J = 8.3, 7.3, H-3ꢀ, 5ꢀ), 7.40 (2H, br.d, J = 8.3, H-2ꢀ, 6ꢀ),
7.50 (1H, d, J = 9.5, H-4). C NMR spectrum (CD OD, ꢅ, ppm): 25.7 (C-8), 28.6 (C-9), 31.6 (C-7), 46.5 (CH ), 51.9
3-4
3
13
4-3
3
3
(C-10), 60.9 (C-13), 62.8 (C-11), 116.4 (C-3), 116.9 (C-5), 120.4 (C-2ꢀ, 6ꢀ), 123.96 (C-4ꢀ), 129.7 (C-3ꢀ, 5ꢀ), 140.1 (C-1ꢀ), 142.9
+
(C-4), 149.9 (C-6), 157.0 (CONH), 164.4 (C-2). HR-MS (EI): [M] 338.1779, calcd for C H N O 338.1737.
15 19
3 2
N,Nꢀ-[Methylene-bis(4,1-phenylene)]dicytisinecarboxamide (9) was prepared from 1 (0.5 g, 2.6 mmol) and
4,4ꢀ-methylenediphenyldiisocyanate (0.27 mL, 1.3 mmol) by the method for 5. Yield of 9, 0.81g (98%), mp 198–199°C
20
–1
(MeOH), [ꢃ] –208.0° (c 1.08, DMSO). IR spectrum (ꢄ, cm ): 3417, 2923, 2351, 2302, 1649, 1595, 1546, 1513, 1418,
D
1
1364, 1343, 1307, 1184, 1157, 1060, 988, 916, 801, 663. Í NMR spectrum (DMSO-d , ꢅ, ppm, J/Hz): 1.89 (1H, m, H -8),
6
syn
2
3
4
2
1.94 (1H, m, H -8), 2.46 (1H, m, H-9), 3.05 (1H, ddd, J = 12.7, J
= 2.5, J
= 1.0, H -11), 3.09 (1H, dd, J = 12.7,
= 1.0, H -10), 3.70 (2H, s, H-7ꢀ), 4.03
anti
11-9
11-10
10-11
exo
3
2
3
4
J
= 2.5, H -13), 3.14 (1H, m, H-7), 3.70 (1H, ddd, J = 15.6, J
= 6.6, J
13-7
exo
10-9
2
exo
2
3
4
3
4
4
(1H, dt, J = 15.6, J
= 1.0, J
= 1.0, H
-10), 4.09 (1H, ddt, J = 12.7, J
= 3.1, J
= 1.7, J
= 1.7, H
-13),
10-9
10-8
endo
13-7
13-11
13-8
endo
2
3
4
4
3
4
4.24 (1H, ddt, J = 12.7, J
= 3.3, J
= 1.7, J
= 1.7, H
-11), 6.21 (1H, d, J = 7.0, J = 1.5, H-5), 6.23 (1H,
11-9
11-13
11-8
endo
5-4
5-3
3
4
3
3
dd, J = 9.1, J =1.5, H-3), 6.98 (2H, d, J = 8.5, H-3ꢀ, 5ꢀ), 7.10 (2H, d, J = 8.5, H-2ꢀ, 6ꢀ), 7.35 (1H, d, J = 9.1, J = 7.0,
3-4
13
3-5
4-3
4-5
H-4). C NMR spectrum (DMSO-d , ꢅ, ppm): 24.9 (C-8), 26.7 (C-9), 33.8 (C-7), 39.6 (C-7ꢀ), 48.5 (C-10), 49.6 (C-11), 50.6
6
(C-13), 105.2 (C-5), 115.5 (C-3), 120.1 (C-2ꢀ, 6ꢀ), 128.3 (C-3ꢀ, 5ꢀ), 135.1 (C-4ꢀ), 137.6 (C-1ꢀ), 139.1 (C-4), 150.1 (C-6), 155.2
15
(CO), 162.4 (C-2). N NMR spectrum (DMSO-d , ꢅ, ppm): 100.92 (NH), 176.17 (N-1). HR-MS (EI): m/z 190, 250, 440.
6
Biological Tests. All compounds were dissolved in DMSO (100%). Stock solutions (1 M in DMSO) were diluted
immediately before the experiments in complete growth medium (DMEM, 10% fetal calf serum, 2 mM L-glutamine,
50 ꢇg/mL gentamicin sulfate) to final concentrations of 1, 10, and 100 ꢇM and DMSO final concentration of 0.1%.
Cytotoxic properties of the compounds were studied using the MTT-test on HEK293 cell line (Russian Collection of
Cell Cultures, Institute of Cytology, RAS, St. Petersburg) according to the manufacturer’s protocol. HEK293 cells (passage
4
7–9) were seeded (2 ꢂ 10 cells per well) in growth medium (100 ꢇL) in 96-well plates. Tested compounds were added at
concentrations of 1, 10, 100, and 1000 ꢇM 24 h after cells were seeded and were incubated for 48 h. MTT reagent was added
4 h before the incubation time expired. Incubation was continued for 4 h. Absorption was measured at 540 nm on EnSpire®
Multimode Plate Readers 2300 (PerkinElmer, USA). The arithmetic averages of normalized absorption values for each
concentration were found. The percent metabolic activity was calculated from the ratio to the control, which was taken as
100%. The IC values (compound concentration required for 50% inhibition of cell viability in vitro) that characterized the
50
501

cytotoxicity were calculated. Results were processed statistically using Microsoft Office Excel 2003 and GraphPad Prism
v.5.0 (GraphPad Software Inc., USA) programs. Results from three independent experiments were expressed as the average
of three measurements for each concentration plus/minus the standard deviation relative to the control (0.1% DMSO) taken as
100%.
The ability of the (–)-cytisine derivatives to regulate the activity of the NFAT-dependent signal cascade was assessed
quantitatively and analyzed comparatively using luciferase reporter constructs [19]. The IC or EC values (compound
50
50
concentration causing luciferase activity to increase by 50%) were calculated by non-linear regression of the logarithm of the
compound concentrations and normalized percent inhibition/activation of luciferase (GraphPad Prism v.5.0, GraphPad Software
Inc., USA).
ACKNOWLEDGMENT
The work was supported financially by the RFBR (Grant No. 12-03-00724-a), the RAS Presidium Basic Research
Program “Fundamental Sciences, Medicine” for 2014, and grants of the RF President for Leading Scientific Schools NSh-
5923.2014.4 and NSh-1700.2014.3.
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